Our work has shown that calcification is normally restricted to skeletal tissue due to the co-expression in bone and cartilage of tissue-nonspecific alkaline phosphatase (TNAP) and fibrillar collagens. TNAP's primary function is to degrade extracellular inorganic pyrophosphate (ePPj), a potent mineralization inhibitor, which is produced ectoplasmically by the enzymatic activity of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and also transported to the extracellular milieu by the action of the ankylosis protein (ANK). Mice deficient in TNAP (Akp2-/-) develop hypophosphatasia characterized by greatly elevated levels of ePPj, which lead to osteomalacia. However, the simultaneous ablation of NPP1 or ANK function, i.e., in [Akp2-/-;Enpp1-/-] and [Akp2-/-;ank/ank] mice, lead to a correction in ePPj levels and an amelioration of osteomalacia. We have also discovered a strict correlation between the levels of ePPj and osteopontin (OPN), another potent inhibitor of mineralization and we uncovered that ePPj upregulates Opn gene expression in osteoblasts. However, in the absence of OPN, mice do not develop osteomalacia but in fact slightly hypermineralize despite them having even larger amounts of ePPi than TNAP-deficient mice. In this competitive renewal application we will focus on testing the central hypothesis that the Pi/PPi-mediated regulation of OPN and the OPN-mediated regulation of Pi/PPi are linked counter-regulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePPi. Our Specific Aims are to test the hypotheses: 1) that OPN controls the expression of the molecules that produce, transport and degrade ePPi and in so doing acts as a feedback mechanism to the ability of osteoblasts to sense and respond to changes in the Pi/PPi ratio;2) that in Opn-/- mice there is a compensatory overproduction of ePPi that serves to avert skeletal pathology. We predict that manipulating ePPi levels upwards will lead to osteomalacia while reducing ePPj levels will cause hypermineralization in Opn-/- mice;3) that it is the combined normalization of ePPi and OPN levels that lead to corrections in calcification in the [Akp2-/-;Enpp1-/- and [Akp2-/-;ank/ank] mice;4) that TNAP plays multiple, interrelated functions in the integrated ePPi-OPN counter-regulatory mechanism including a) contributing to the Pj pool, b) contributing to the ePPi pool by both degrading and producing PPj, and thus competing with the function of NPP1 and c) modulating the biological function OPN through dephosphorylation. Our work will clarify the synergistic and hierarchical role of PPj and OPN as inhibitors of calcification in health and disease and the mechanisms by which bone-forming cells are able to respond to changes in the concentrations of these inhibitors to maintain physiological homeostasis.